Detection of lysergic acid diethylamide, delta-9-tetrahydrocannabinol and related compounds by plasma chromatography. Plasma chromatography as a method for ultratrace qualitative and quantitative detection of organic compounds is especially well suited for detection of gas chromatographic effluents. The optimum range of sample quantity is 10-6 to 10-12 g for detection and identification of a compound by use of its characteristic positive and negative mobility spectra. The type of reference mobility spectra produced by alkanes, aromatics, esters, halogenated compounds, nitrogenated compounds and organic acids have been previously reported. This study presents the reference mobility spectra produced for lysergic acid diethylamide (LSD), delta-9-tetrahydrocannabinol (delta-9-THC), digitoxigenin and several biochemical compounds of research significance. LSD and delat-9-THC in a mixture can be detected and identified by plasma chromatography positive mobility spectra in quantities of 10-7 g or less. All the compounds investigated in this study display strong MH-+ ions along with other ions primarily of the type (M)NO-+, (M)2H-+. None of these compounds exhibits negative mobility spectra.


A simple gas-liquid chromatographic method with electron capture detection for the determination of progesterone in the blood of humans and domestic animals. A simple routine method is reported for the determination of progesterone in blood by gas-liquid chromatography. An unlabelled internal standard, testosterone acetate, was added to plasma samples. Following preliminary purification by thin-layer chromatography, progesterone in pregnancy plasma was evaluated as the free steroid with a flame ionization detector. The hormone in the plasma of women during the menstrual cycle and of cycling domestic animals was determined as the 3-enol ester heptafluorobutyrate by electron capture detection. The validity of testosterone acetate as an internal standard was proved by simultaneous processing of [7alpha-3H]progesterone and a [4-14C]testosterone acetate and the determination of the isotope ratio in samples. The results of control experiments and normal values are also presented.


Gel chromatography of steroid oestrogens on sephadex LH-20. The behavior of 23 steroid oestrogens Sephadex LH-20 using six solvent systems was investigated. The fractions eluted by gel column chromatography were analysed by thin-layer chromatography and spectrometry. The method was used for the radiochemical purity of labelled compounds by isotopic dilution.


Isolation and separation of tall fescue and ryegrass alkaloids. Bases were extractable from dried, powdered grass material by percolation with methanol. The bases were removed from the methanol by cation-exchange resins and subsequently removed from the resins. Isolation of bases by these methods eliminated the problems associated with the use of large quantities of chlorinated solvents and formation of emulsions found in previously described isolation methods. The bases were separated by paper and thin-layer chromatography (TLC). TLC and a spectrodensitometer were used to quantify the principal alkaloids in crude extracts of tall fescue samples. This procedure recovered all of the alkaloids in spiked samples, and the standard deviation for perlolidine and perloline in tall fescue samples was 3% or less of the mean value.


Quantitative gas chromatographic determination of nitrilotriacetic acid in the presence of other carboxylic acids. Nitrilotriacetic acid was separated (as its tri-n-butyl ester) from twenty-four other fatty, phenolic and polycarboxylic acids, using prepacked commercial OV-210 columns. Quantitative analysis down to 0.010 mg nitrilotriacetic acid per liter was demonstrated. The molar responses and retention times of the acids, relative to phthalic acid, were determined.


Gas chromatographic determination of micro-amounts of cyclamates. It was found that triethylammonium cyclamate is converted into N-hepta-fluorobutyrylcyclohexylamine in a high and constant yield by reaction with hepta-fluorobutyric anhydride at 90 degrees for 1 h, and gas chromatography of the product gives a sharp peak that is highly sensitive to an electron capture detector. A useful method for the micro-determination of cyclamates was established by combining this reaction with gas chromatography.


Quantitative assay of diclofenac in biological material by gas-liquid chromatography. Diclofenac and an internal standard are isolated from biological samples by acid-specific extraction and converted into the corresponding indolones with 0.5% sulphuric acid in 2,2,2-trifluorethanol. The neutral derivatives are isolated by solvent extraction and subjected to gas-liquid chromatography. The use of an electron capture detector permits the assay of diclofenac at levels down to 2 ng per sample.


[Determination of chloroquine in biological material by gas-liquid chromatography (author's transl)]. A method is developed for the gas chromatographic determination of chloroquine after extraction from biological material, using medazepam as the internal standard and a column filled with 3% OV-17 on Gas-Chrom Q. The detection limits are: 0.15 mug/ml of urine, 0.25 mug/ml of whole blood, and 1.50 mug/g of tissue. The technique can be applied in toxicological analysis and in monitoring drug levels.


A gas chromatographic method for measuring 6-mercaptopurine in serum. A method is presented for the measurement of 6-mercaptopurine (6-MP) in serum. Serum samples containing the drug were treated with dithioerythritol and saturated with ammonium carbonate. 6-MP was then extracted from the serum with a mixture of isopropanol-ethyl acetate (1:1, v/v) containing 0.01% ethanethiol. The solvent was evaporated, the residue dissolved in dilute hydrocholoric acid, washed with chloroform, and extracted with ethyl acetate. 6-MP was derivatized with trimethylanilinium hydroxide and measured gas chromatographically. The sulfhydryl-protecting reagents, dithioerythritol and ethanethiol, were added to prevent the decomposition of 6-MP. The extraction and clean-up method recovered 78 +/- 2% (S.E.) of the 6-MP present. Tracer amounts of [8-14C]6-MP served as the internal standard during the extraction part of the method. Theophyline was used as the internal standard during the gas chromatographic analysis. The standard curve obtained from the gas chromatograph was linear between 0.5 and 20 mug/ml of 6-MP. Serum samples were stored in the freezer for two weeks without significant loss of drug. No interference was encountered from normal serum constituents or xanthines, such as caffeine or theophylline added to serum.


Gas chromatographic-mass fragmentographic determation of "steady-state" plasma levels of imipramine and desipramine in chronically treated patients. A sensitive and specific method for the simultaneous determination of imipramine and desipramine in the blood plasma of depressed patients under treatment that involves separation by gas chromatography and detection by mass fragmentography is described. Concentrations were determined by focusing the mass spectrometer upon the ions at m/e 280 and 235 for imipramine and 308, 236 and 114 for desipramine (N-acetyl derivative), while promazine was used as the internal standard (ions at m/e 284 and 238). Determinations are possible at levels as low as 10 ng/ml in plasma.


Gas chromatographic confirmation of amino acid structure via diastereomer preparation. The partial gas chromatographic (GC) resolution of several amino acid N-trifluoroacetyl 2-butyl ester diastereomeric derivatives on a short packed column has been used to confirm GC peak assignments in analysis of a marine sediment sample.


Electron-capture gas chromatography of methadone after oxidation to benzophenone. A procedure for the determination of low levels of methadone (6-dimethylamino-4,4-diphenylheptanone-3) in serum has been developed. Methadone is extracted from serum into n-heptane and re-extracted into an acidic aqueous phase. Methadone is oxidized to benzopheone with barium peroxide in sulphuric acid, during which procedure an n-heptane phase is present into which the oxidation product is continuously extracted. The benzophenone formed is determined by means of electron-capture gas chromatography. The recoveries are 100 +/- 3% and 100 +/- 4.5% at the 120 and 16 ng levels, respectively. The minimum amount that can be determined in 1 ml of serum is 4 ng. Interferences from possible metabolies are probably minor. The main cyclic metabolite is only co-determined to a minor extent if the oxidation time is optimized. Comparison of this oxidation method with a combined gas chromatographic-mass spectrometric determination with selected ion monitoring showed identical serum levels.


Gas chromatographic determination and gas chromatographic-mass sepectrometric analysis of chloramphenicol, thiamphenicol and their metabolites. A rapid and accurate gas chromatographic (GC) method is described for the determination of chloramphenicol, thiamphenicol and their metabolites excreted in human urine. These excretions were pre-treated with diazomethane and N,O-bis(trimethylsilyl)acetamide, so that they could be easily subjected to GC with satisfactory separation from each other and also from other urinary excretions, and could be determined simultaneously. The structures of the metabolites were confirmed by GC combined with mass spectrometric measurements of the GC peaks. The application of the method to urine specimens enabled more precise results for the amounts of metabolites present to be obtained; the excretion of thiamphenicol glucuronide was not observed.


Combined ultraviolet-fluorescence detection in high-pressure liquid chromatography of pharmaceuticals. The use of combined UV-fluorescence detection for the evaluation of incompletely resolved compounds and trace components in the presence of large quantities of major components is described, analysis for thioridazine and some of its oxidation products by high-pressure liquid chromatography being chosen as a practical example. Mesoridazine and the ring oxide of thioridazine have been determined quantitatively with relative standard deviations (n = 6) of 2.0 and 3.6%, respectively, at concentrations below 0.1 mug per injection. Resolution of the two components is difficult and, in this instance, unnecessary. By a similar approach, it was possible to determine the highly fluoresecent sulforidazine at a concentration of 0.4% of the thioridazine with 6.2 mug of thioridazine injected. A relative standard deviation of 5% was attainable at this concentration. Fluorescence detection limits for mesoridazine and sulforidazine at a signal-to-noise ratio of 4:1 are between 5 and 10 ng per injection; this corresponds to about 0.1% of the active substance for the above example.


Agarderivatives for chromatography, electrophoresis and gel-bound enzymes. IV. Benzylated dibromopropanol cross-linked sepharose as an amphophilic gel for hydrophobic salting-out chromatography of enzymes with special emphasis on denaturing risks. The preparation of benzylated covalently cross-linked Sepharose 2B is described. Such gel was analyzed for its degree of substitution, and gels with three different degrees of substitution were used in chromatographic experiments with dextranase, alpha-amylase, lactate dehydrogenase, alpha-chymotrypsin and trypsin. Yields and chromatographic patterns for different eluting systems were determined. It was found that gradients combining an increase in ethylene glycol concentration with a decrease in salt concentration gave better results than did pure salt gradients. No denaturation was observed for dextranase or alpha-amylase, but the other enzymes tested were partly denatured. The most severe denaturation was observed for lactate dehydrogenase desorbed from the highest substituted gels, although the enzyme was highly active in the adsorbed state. The results and the use of amphophilic gels are discussed.


Determination of nitrazepam in serum by gas-liquid chromatography. Application in bioavailability studies. A gas chromatographic method with electron capture detection has been developed for the analysis of nitrazepam in serum. N-Desmethyldiazepam is used as internal standard. Nitrazepam isolated from serum is converted by acid hydrolysis into 2-amino 5 nitrobenzophenone, which is chromatographed. Metabolites of nitrazepam (7-amino and 7-acetamido compounds) are not included in the determination. Recovery experiments showed that the method is quantitative. The limit of detection is 5 ng/ml of nitrazepam in serum. The method has been used for measuring serum concentrations of nitrazepam in bioavailability studies on subjects given a single dose of nitrazepam tablets.


Controlled pore glass chromatography of protein-sodium dodecyl sulphate complexes. The inclusion of urea has been found to eliminate adsorption of protein-sodium dodecyl sulphate (SDS) complexes to controlled pore glass. Using buffer containing 6 M urea, 0.5% SDS and glass with pore diameter 12.3 nm, it is possible to determine protein molecular weights in the range 3500-12,000. Results with glass of larger pore diameter (25.5 nm) are similar to those reported in the absence of urea in the molecular-weight range 12,000-140,000. Controlled pore glass chromatography also permits the study of the relative importance of conformation free of charge effects for those proteins which deviate from the normal calibration curve for SDS-polyacrylamide gels.


Separation of the methylated nucleoside consituents of ribonucleic acid by two-dimensional thin-layer chromatography. In an attempt to separate a number of methylated nucleoside constituents reportedly present in various mammalian RNA preparations, a comparison of the migration of selected nucleoside standards was assessed via thin-layer chromatographic techniques with respect to three solvent systems and an adsorbent of varying proportions of silica gel and microcrystalline cellulose. The results of these studies revealed that a mixture of seventeen methylated constituents could be separated following two-dimensional development on an adsorbent containing 60% cellulose and 40% silica gel. Constituents separated included the 2'-O-methylated derivatives of adenosine, guanosine, cytidine and uridine, 1-, N6- and N6,N6-methyladenosines, 1-methylinosine, 1-, 7-, N2- and N2,N2-methylguanosines, 3-, 4- and 5-methylcytidines and 3- and 5-methyluridines. In addition, all of the above constituents were separated from their respective non-methylated nucleosides and bases.


The direct gas chromatographic analysis of barbiturates as their sodium salts. The addition of formic acid to the carrier gas will permit the on-column liberation of the free acids following the direct injection of barbiturates as their sodium salts. These free acids may then be resolved upon an Apiezon L column: the presence of formic acid also minimizes the adsorption effects which may be associated with the gas chromatographic analysis of these relatively polar drugs.


Rapid analysis of individual tablets by high pressure reverse phase liquid chromatography. A high pressure liquid chromatographic determination of a steroidal oxime in the pharmaceutical dosage form is reported. The reversed phase liquid-liquid partition chromatography employing ODS-Permaphase as support allows thirty to forty individual tablets to be analyzed per hour at a dose level of 50 micrograms to 1 milligram per tablet. The method is simple, rapid, accurate and sensitive.


Gas chromatographic evaluation of the volatile constituents of lung, brain and liver tissues. Volatile metabolites from rat liver, lung and brain tissues were compared using gas chromatography. Volatiles released from the homogenized tissues at 95-100 degrees C were collected on a poly phenyl ether solid adsorbent. The adsorbed volatiles were examined by high-resolution gas chromatography. Markedly differing overall volatile profiles were observed for the tissue types examined, and it appears that certain constituents may be characteristic of a particular tissue.


The examination of some vinyl acetate/olefin copolymers by pyrolysis gas chromatography mass spectrometry. A study of the thermal degradation of some vinyl acetate/olefin copolymers reveals that side chain scissions at the backbone and scissions within the side chains themselves yield fragments which are apparently characteristic of the parent olefin.


Structural analyses of triglycerides containing isotopically-labelled fatty acids. The stereospecific analysis of triglycerides is a complex, multistep procedure and some losses inevitably occur at every stage. Such losses are particularly important when the triglycerides contain isotopically-labelled fatty acids incorporated by incubation in vitro with the relevant enzyme systems, but they can be compensated for by adding, prior to the analysis, a triglyceride containing only the same labelled odd-chain fatty acid in each position.


Determination of plasma lipid profiles by automated gas chromatography and computerized data analysis. Plasma or serum [ 0.1-1.0 ml] was digested with phospholipase C and total lipid extracts were prepared and silylated in the presence of tridecanoylglycerol as internal standard. The neutral lipid and free fatty acid profiles were determined by means of an automated GLC system equipped with an unheated on-column inlet, time actuated liquid injector, programmed heating, cooling and equilibration cycles, and an electronic peak area integrator. The separations were accomplished on a 50 cm x 2 mm i.d. steel column packed with 3% OV-1 on100-120 mesh Gas Chrom Q using nitrogen as a carrier gas in the temperature range 175-350 degrees C. The tube number, peak retention time and peak area were recorded on a punched paper tape, which was subsequently read into a computer via a time-share terminal. The composition of the sample was calculated in relation to the internal standard using a modification of a commercially available computer program and the results were expressed as mg or mole % and characteristic molar ratios of lipid classes. In addition to estimates for total cholesterol and triglyceride, the method provides a detailed account of individual or small groups of molecular species of various lipid classes, which is a major advantage over other automated methods of plasma lipid analyses.


Effect of paramethasone acetate on ovarian steroids and gonadotropins. I. Normal menstrual cycle. In an attempt to elucidate the effect of paramethasone acetate on the hormonal profile of the normal menstrual cycle, 5 clinically healthy women, aged 24-36 yr, with ovulatory cycles were studied during a control menstrual cycle and during treatment with paramethasone acetate (6 mg/day). The length of the untreated cycle was 29.2 +/- 2.3 days as compared to 29.0 +/- 3.1 days during the paramethasone-treated cycle. Peak plasma E-2 level was 188.2 +/- 42.1 pg/ml in the control cycle and 74.4 +/- 15.3 pg/ml during paramethasone treatment (P is less than 0.05). Peak plasma 17 OH-P was 4.0 +/- 0.4 ng/ml in the control cycle and 1.6 +/- 0.2 ng/ml during the paramethasone treated cycle (P is less than 0.005). No significant differences in plasma progesterone were observed during the luteal phase of both cycles. Minimal and scattered differences were observed in plasma FSH. However, plasma LH levels were lower in the paramethasone acetate than in the control cycle almost throughout the entire period of study. Furthermore, midcycle LH peak in the control cycle was 958 +/- 104 ng/ml as compared to 283 +/- 24 ng/ml during the paramethasone treatment (P is less than 0.005). Despite these differences, ovulation occurred during paramethasone treatment based upon the observed rise in BBT, the plasma progesterone levels above 6 ng/ml and the secretory changes in the endometrium. These results suggest that: 1) Paramethasone may block E-2 synthesis at the ovarian level and , 2) Ovulation may still occur even in the presence of E-2 and LH plasma concentrations lower than those occurring in the normal menstrual cycle.


The significance of partial suppressibility of serum thyroxine by triidothyronine administration in euthyroid man. Recent evidence indicates that triiodothyronine (T3) administration may not completely inhibit normal thyroid secretion. To further corroborate this observation, measurement of serum T4-RIA concentrations was performed on 15 normal controls (10 men, 5 women; ages 20-42) who were placed on 100 mug of T3 daily for a 5-week period. Decrements of 53%, 36%, and 28% from the baseline T4-RIA were noted at weeks 1, 2, and 3 respectively. At 3 weeks a nadir T4-RIA of 2.5 mug/100 ml was reached which did not significantly differ from the 4th (2.9 mug/100 ml) and 5th weeks (2.6 mug/100 ml). Further, seven euthyroid patients who had received replacement thyroid hormone for 1-16 were switched to T3 (75-100 mug/day) for 28 days. At the end of this period, their mean T4-RIA was 2.6 mug/100 ml. Similar T3 treatment studies were performed on 20 primary hypothyroid patients. After 4 weeks of T3 all 20 patients displayed a T4-RIA below the limits of assay detectability (less than 0.625 mug/100 ml) while all euthyroid subjects had values greater than 1.2 mug/100 ml. Suppression of T4-RIA with T3 was also noted in 4 patients with pituitary and 2 patients with hypothalamic hypothyroidism. Three days after cessation of T3 treatment in normal subjects, no significant rise in mean T4-RIA was seen (2.3 mug/100 ml). Subsequently, T4-RIA rose to 4.5 mug/100 ml on day 7 and 6.7 mug/100 ml on day 10 (74% of the presuppression value) in normals. A similar rise to 7.9 mug/100 ml 10 days after withdrawal from T3 was noted in the euthyroid subjects who had received long-term thyroid hormone replacement. In contrast, all primary hypothyroid patients had either a minimal or nondetectable elevation in T4-RIA while demonstrating a marked rise in TSH 10 days after T3 withdrawal. An absent or impaired rise in T4-RIA after T3 withdrawal was also noted in patients with pituitary and hypothalamic hypothyroidism. These observations indicated: 1) There is continued thyroidal T4 secretion in euthyroid subjects receiving 100 mug of T3 daily. 2) The hypothesis is advanced that an intact hypothalamic-pituitary-tyhroid axis may be required for continued T4 secretion while on T3. 3) The duration of prior suppression with thyroid hormone medication does not appear to influence this phenomenon.


Cushing's syndrome with fluctuation due to adrenal adenoma. A case of fluctuating Cushing's syndrome due to an adrenal adenoma is described. Plasma corticosteroids were frequently low and urinary steroids fluctuated markedly over a 15-month period. Only the response to dexamethasone was consistently abnormal and indicative of Cushings's syndrome.


A new method simplifying collection of serial specimens for gonadotropin determinations. Patterns of gonadotropin levels have been compared in specimens of serum, 24-h urine collections, and first morning urine voidings collected throughout a menstrual cycle from 8 sexually mature, young women. Quantities of gonadotropins in first morning voidings were found to provide a reliable estimate of 24-h excretion of these hormones and demonstrate the same patterns of change as do serum and 24-h urine specimens. Thus first morning voidings satisfactorily overcome logistical problems inherent is serial serum and 24-h urine sampling for studying gonadotropin excretion patterns.


A radioimmunoassay for prostaglandin A1 in human peripheral blood. A specific, sensitive and accurate radioimmunoassay (RIA) method for the measurement of prostaglandin A1 (PGA1) in either human whole blood or plasma is described. Whole blood is immediately lysed with distilled water containing tritiated indicator. When plasma is assayed, the blood samples are handled at 4 C and rapidly centrifuged. The lysate or plasma is adjusted to pH 5 with buffer and quickly extracted with 5% methanol in dichloromethane. The whole blood or plasma extract is then purified by Sephadex LH20 chromatography using the system methanol: methylene chloride (5:95) which separates the major groups of PGA, PGE and PGF. The RIA is then performed using an antiserum generated in rabbits from PGA1 coupled to bovine thyroglobulin. The antibody is highly specific, possessing very low cross reactivity to other prostaglandins (PGA2, PGE, PGB and PGF). Activated florisil or ammonium sulfate can be used to separate bound from free prostaglandin. This whole blood or plasma method yields blank values of only 2 +/- 2 pg per sample with a between assay precision determined by duplicate analysis of 8% and interassay precision of 3%. The mean whole blood PGA1 concentration in 27 subjects in 2.5 +/- 1.6 (SD) ng per 100 ml. No significant sex difference in PGA1 levels was noted and values were similar whether measured in whole blood or cooled plasma rapidly prepared and extracted. These values of PGA1 are much lower than those RIA values reported by others for "PGA" using antibodies with lower specificities.


Thyroglobulin biosynthesis in "cold" and "hot" nodules in the human thyroid gland. Studies on "cold" and "hot" nodules from human thyroid gland have been carried out to investigate thyroglobulin biosynthesis and to correlate carbohydrates incorporation and thyroid hormone formation in thyroglobulin. The aim of the investigation was to ascertain the role of carbohydrates of the protein molecule in its migration to the iodinating site of the cell. Thyroid slices from two cases with "cold" and two with "hot" nodules were incubated for 30 to 120 min with [3H]leucine, [3H]carbohydrates (ManNAc, Gal, Man, GlcNAc, GalNAc) and 125I. Soluble and particulate iodoproteins, solubilized by digitonin, were identified by density gradient centrifugation and immunoprecipitation; following hydrolysis carbohydrates and iodothyronines were identified by paper chromatography and chemically determined. While the rate of leucine incorporation into TG increased with time in both "cold" and "hot" nodules, the "cold" nodule, with respect to the "hot" tissue, showed: much lower efficiency incorporating carbohydrates and iodine into TG; lower thyroid/medium ratio of radioiodine; less total soluble protein and soluble TG; more particulate protein and solubilized TG;  1/3 of the carbohydrate and iodine chemical content; only minute amounts of labeled iodothyronines in TG compared to normal levels in "hot" tissue. The formation of thyroid hormone, in enzymatically iodinated TG, and the iodinating activity of the 105,000 X g pellet, incubated with low iodine TG, were similar in both "cold" and "hot" tissues. These results demonstrate that the "cold" nodule is able to synthesize thyroglobulin but the protein is defective in its carbohydrate content. The deficient iodine transport into "cold" tissue and impaired hormonal synthesis are confirmed. The incomplete incorporation of carbohydrates into thyroglobulin in the "cold" nodule could be important for the maturation and migration of the molecule to the iodinating site of the cell


Gastric inhibitory polypeptide (GIP) stimulated by fat ingestion in man. Ten normal volunteers ingested emulsified corn oil and the immunoreactive GIP, insulin (IRI) and nonesterified fatty acid (NEFA) responses were measured. Serum GIP levels increased after the ingestion of corn oil in all subjects, rising from a mean fasting level of 272 pg/ml to 856 +/- 272 pg/ml (P less than 0.05) by 30 minutes. The peak mean serum GIP  concentration of 1,345 +/- 291 pg/ml occurred at 60 minutes; and mean serum GIP levels at 180 minutes remained significantly elevated over fasting values. Serum IRI, glucose and NEFA concentrations did not change during the 180 minutes of study. No changes in serum GIP concentrations occurred when, for control purposes, six volunteers ingested water on another day. We conclude: 1) Fat is a potent stimulus for the release of GIP in normal individuals. 2) Endogenously released GIP is not insulinotropic under the conditions of this study.


Determination of common parameters fo iodothyronine metabolism and distribution in man by noncompartmental analysis. A noncompartmental method is proposed for the calculation of the total distribution volume of exchangeable iodothyronine, the fractional irreversible removal rate from the exchangeable pool, and the metabolic clearance rate of labeled triiodothyronine (T3) and thyroxine (T4). The method depends on a graphic analysis and does not require the use of computers. The results of noncompartmental analysis of T3 and T4 distribution and metabolism are compared to single compartmental calculations in 12 subjects. Single compartmental analysis appeared to overestimate the distribution volume of T3 by a factor of 2.9, the metabolic clearance rate by a factor of 1.4, and underestimate the fractional removal rate by a factor of 0.48. Whereas single compartmental analysis did not introduce a significant error into the calculation of the metabolic clearance rate of T4, the distribution volume appeared to be overestimated by a factor of 1.4 and the fractional removal rate underestimated by a factor of 0.9.


Pharmacodynamics of propylthiouracil in normal and hyperthyroid subjects after a single oral dose. Serum levels of propylthiouracil were measured in 8 normal persons and in 7 patients with hyperthyroidism after a single, 300 mg, oral dose of 6-n-propyl-2-thiouracil (PTU). The patients with hyperthyroidism were restudied after 3, 6, and 9 weeks of individualized treatment with PTU. The serum half-life of the drug in normal subjects was 1.65 h. In patients with hyperthyroidism the serum half-life was similar, and it did not change significantly as the euthyroid state was achieved.


Disposition of a new, nonsteroid, antiandrogen, alpha,alpha,alpha-trifluoro-2-methyl-4'-nitro-m-propionotoluidide (Flutamide), in men following a single oral 200 mg dose. A single oral 200 mg dose of tritium-labeled alpha,alpha,alpha-trifluoro-2-methyl-4'-nitro-m-propionotoluidide (flutamide) was given to 3 men. Analysis of plasma, urine and feces shows flutamide is rapidly and completely absorbed and excreted mainly through the kidneys. Analysis of plasma radioactivity shows flutamide is rapidly and extensively metabolized--only 2.5% of plasma radioactivity 1 h after dosing is associated with flutamide. At least 10 other metabolites are present, of which 6 have been tentatively identified. These are alpha,alpha,alpha-trifluoro-4'-amino-m-acetotoluidide (A); alpha,alpha,alpha-trifluoro-4'-amino-2-methyl-m-lactotoluidede (B); alpha,alpha,alpha-trifluoro-4'-nitro-m-acetotoluidede (C); alpha,alpha,alpha-trifluoro-2-methyl-4'-nitro-m-lactotoluidide (D); alpha,alpha,alpha-trifluoro-4'-amino-2-methyl-m-propionotoluidide (E); and alpha,alpha,alpha-trifluoro-6-nitro-m-toluidine (F). (D) represents 23% of plasma tritium 1 h after drug and is a major metabolite at all other measured times. Each of the other metabolites accounts for less than 10% of plasma radioactivity. Minor amounts of flutamide and (A) through (F) are found in 0-24-h urine. An additional metabolite, alpha,alpha,alpha-trifluoro-2-amino-5-nitro-p-cresol, constitutes 25% of urine tritium. The rapid conversion of flutamide to (D) and the high plasma levels of (D) suggest (D) might be the active form of flutamide.


The effect of chlorpromazine on the secretion of immunoreactive beta-MSH and prolactin in man. The effect of chlorpromazine (50 mg. im) on the plasma concentration of immunoreactive beta-melanocyte-stimulating hormone (beta-MSH) and prolactin was studied in 8 hospitalized subjects with non-endocrine skin disorders. Plasma beta-MSH concentrations remained unchanged over a period of 7 h in 6 subjects. In the remaining 2 subjects there was a slight increase. Plasma prolactin concentrations were greatly increased in all subjects 1 1/2-3 h after the injection and had almost returned to pre-injection levels by 7 h. This suggests that the control of beta-MSH secretion in man, unlike that of prolactin in man and MSH peptides in other mammals, is not predominantly inhibitory. The reason for this discrepancy may be that beta-MSH is not a natural MSH in man and occurs as part of the lipotropic hormone (LPH) or as a breakdown product.


Failure of oral water loading and intravenous hypotonic saline to suppress plasma prolactin in man. To test the effect of acute changes in plasma osmolality on plasma prolactin concentrations, the hormone was measured before and during oral water loading, hypotonic saline infusion, and hypertonic saline infusion in normal subjects. In 10 normal men there was a small but statistically significant rise in mean prolactin, from 7.6 to 12.3 ng/ml, occurring within half an hour after the ingestion of a water load of 20 ml/kg. A small but statistically insignificant rise in mean plasma prolactin was observed in 11 normal women, although there was a significant negative correlation between plasma prolactin and plasma osmolality in these subjects. No effect of the intravenous infusion of either hypotonic saline (0.45%) or hypertonic saline (5%) on mean plasma prolactin was noted in 5 normal men. These studies do not confirm a previously reported suppression of prolactin concentrations by oral water loading of hypotonic saline infusions in normal subjects. While the data suggest a negative correlation between plasma osmolality and plasma prolactin, at least after water loading, they do not support a physiological role for prolactin in the short-term regulation of plasma osmolality in humans.


Ewing's sarcoma as a cause of the syndrome of inappropriate secretion of antidiuretic hormone. A patient with Ewing's sarcoma presented with the syndrome of inappropriate secretion of antidiuretic hormone (SIADH) (1). Plasma values for vasopressin were found to be over four times the normal values expected for the plasma osmolality. At postmortem examination, the arginine vasopressin concentration in the tumor tissue was ten times that of the plasma. These data suggest that Ewing's sarcoma may cause SIADH.


Disappearance rates of plasma growth hormone after intravenous somatostatin in renal and liver disease. Plasma immunoreactive growth hormone (hGH) was measured before, during and after the administration of intravenous somatostatin to 3 patients with chronic renal failure and 4 with severe liver disease who had elevation of basal hGH. During somatostatin infusion, the hGH levels declined acutely in a linear fashion when log hGH was plotted against time. Rather surprisingly, the plasma hGH half disappearance time (t 1/2) was 27 min and 18 min in liver and renal disease respectively. These values do not differ from data obtained on normal subjects using exogenous hGH, labelled or unlabelled. Control data on normal subjects using this technique are not available as it was not possible to measure subnormal levels of plasma hGH with the required precision. It is possible that our findings of plasma hGH T 1/2 in liver and renal disease within the normal range reported using exogenous hGH might suggest that high levels of plasma hGH found in these two diseases are primarily caused by hypersecretion rather than impaired clearance.


Endometrial progesterone concentrations during the menstrual cycle. Endometrial progesterone concentrations have been determined during the menstrual cycle and compared to plasma progesterone and estradiol-17 beta concentration. There is a good relationship between endometrial and plasma progesterone concentrations but none between endometrial progesterone and estradiol-17 beta concentration.


Altered hypothalamic-pituitary-adrenal responsiveness to dexamethasone-insulin tolerance test in active acromegaly. Eight acromegalic patients showed a plasma cortisol (11-OHCS) rise after insulin hypoglycemia which was similar to that seen in control patients, with mean peak values (+/-SEM) of 23.2 +/- 3.5 mug/100 ml and 27.2 +/- 3.3 mug/100 ml, respectively. One mg of dexamethasone was given the evening prior to repeat insulin hypoglycemia (DEX-ITT). After dexamethasone, the control subjects showed a mean post hypoglycemic plasma 11-OHCS rise to 18.3 +/- 2.3 mug/100 ml. In contrast, acromegalic patients had a negligible rise is plasma 11-OHCS, despite a comparable degree of hypoglycemia. These data indicate that, in active acromegaly, abnormal hypothalamic-pituitary-adrenal suppressibility can be induced to  insulin hypoglycemia after dexamethasone.


Big growth hormone (GH): conversion to small GH without peptide bond cleavage. Mercaptoethanol treatment of big growth hormone (GH) obtained from human pituitary resulted in a 60% conversion to small GH. Further dissociation was obtained by combined treatment with the reducing agent plus urea. The results indicate that the existence of big GH is dependent upon the formation of inter-polypeptide chain disulfide bonds.


Metabolic clearance and blood production rates of estradiol in hyperthyroidism. The metabolic clearance rate of 17beta-estradiol (MCR2), the plasma levels of 17beta-estradiol (E2)1, sex-steroid binding globulin (SSBG), luteinizing hormone (LH) and follicle-stimulating hormone (FSH) were measured in 10 hyperthyroid subjects (7 men and 3 women). The blood production rate of 17beta-estradiol (PB2) was calculated for all subjects. Nine of the 10 hyperthyroid subjects had a decreased MCR2 which returned towards normal in 5 of the 6 subjects restudied following therapy. In all 10 subjects the levels of SSBG were increased when they were hyperthyroid and returned toward normal with therapy. It is concluded that the decrease in MCR2 is largely due to the increased binding of 17beta-estradiol to SSBG. In 7 of the 10 hyperthyroid the plasma E2 concentrations were normal whereas 3 had slightly elevated levels. In 8 of the 10 hyperthyroid the PB2 was within the normal range. Only 2 hyperthyroid subjects had slightly elevated PB2. In the 6 subjects who were restudied after therapy, there was no consistent change in PB2 which remained in the normal range in all cases. It is concluded that the MCR2 is decreased in most subjects with hyperthyroidism in association with an increase of SSBG. Despite this change in MCR2 there is no significant change in PB2. The increase in SSBG levels in hyperthyroidism appears to be a direct effect of the elevation of thyroid hormone activity and is not mediated through estrogen.


Acute-phase insulin secretion and glucose tolerance in young and aged normal men and diabetic patients. A defect in the early phase of insulin secretion has been proposed as a major determinant of the impaired glucose homeostasis of adult onset diabetes. Since the process of aging is also associated with a deterioration of glucose tolerance, we have measured the acute phase of insulin secretion and glucose disposal rate (KG) after a 20 g glucose pulse in 11 normal, healthy men greater than 65 yr and compared it with the response in 11 nondiabetic men 20-31 yr. The mean fasting plasma sugar levels of the aged subjects (107 mg/dl), tended to be higher than that of the young (94 mg/dl). Comparison of Kg provided clear separation (P less than 0.01) of the old (mean Kg = 0.93) from the young population (mean Kg = 1.95). The acute insulin response to the glucose pulse when expressed either as the area above basal for the 10 min post stimulation or as the mean encremental increase above basal at 3, 4 and 5 min (delta 3-5 IRI) was the same in both timing and magnitude for the old and young subjects. Although our aged subjects had clearly abnormal kg's their acute phase of insulin secretion in response to glucose was normal. Seven noninsulin requiring diabetics with fasting plasma glucose levels greater than 140 mg/dl similarly studied had abnormal kg's (mean = 0.55) but markedly attenuated early release of insulin. Therefore, even though in the diabetics a deficiency of acute phase insulin secretion may have contributed to their impaired kg, in our aged subjects the lack of correlation between kg and a deficiency of acute phase insulin release suggests that factors other than an impairment in the early phase of insulin secretion are responsible for their glucose intolerance or, that there is a dissociation between the biologic and immunologic activity of insulin in these aged subjects.


Treatment of Cushing's disease with reserpine and pituitary irradiation. Eighteen patients with Cushing's disease were treated with reserpine and pituitary irradiation. Complete remission was obtained in 9 out of 18 patients after reserpine treatment of 1-2 mg per day for a mean period of 20.4 months, and pituitary irradiation with a mean of 5,865 rads. In another 9 patients, reserpine 0.8-2 mg per day for a mean period of 22.5 months, and pituitary irradiation with a mean of 6,650 rads, were employed. Of these 9 patients, an additional subtotal adrenalectomy was carried out in 6 patients who are now in complete remission. Because of severe psychic symptoms resulted from the original disease in 2 of the remaining 3 patients, subtotal adrenalectomy was performed first and pituitary irradiation and reserpine treatment followed. Remission was eventually obtained in these 2 cases. One patient refused the operation, and thus had little clinical remission. All of the 17 cases in remission were followed up for periods of 6 months to 10 yr. During this time, only one case which had responded to reserpine and pituitary irradiation relapsed, but regained remission following resumption of therapy. Another died of cerebral glioblastoma 4 yr after remission of the disease. It was noteworthy that endocrinologic data including: plasma levels of ACTH and 11-OHCS, suppressibility by dexamethasone, responses of plasma GH to arginine and to insulin loads, and diurnal rhythm of plasma 11-OHCS were nearly normal in a considerable number of the cases in remission. Effectiveness of the combined therapy with reserpine and pituitary irradiation for treating Cushing's disease may support a working hypothesis that reserpine acts through some as yet unknown mechanism to correct a presumed central nervous disorder, while suitable pituitary irradiation probably corrects the pituitary dysfunction directly.


The inhibitory effect of somatostatin on growth hormone, insulin and glucagon secretion in diabetes mellitus. The inhibitory effect of somatostatin on insulin, glucagon and growth hormone secretion was studied in 5 patients with diabetes mellitus. In three maturity onset diabetics, somatostatin infusion abolished the insulin rise induced by breakfast and oral glucose, and in 2 of them, inhibited the basal insulin secretion by 50% seen during control studies. Concomitantly, there was a marked and prompt reduction of glucagon levels (50%) with a sustained effect. The plasma glucose levels were either unchanged or slightly increased. Following the somatostatin infusion, there was a prompt rebound increase in both insulin and glucagon levels with a relatively stable plasma glucose concentration. In contrast, a drastic reduction of plasma glucose in face of a relatively small fall in plasma glucagon in response to somatostatin infusion was observed in 2 insulin-dependent diabetics. In all patients, the episodic release of growth hormone seen during the control day was abolished during somatostatin infusion.


Plasma norepinephrine and epinephrine in acromegaly. Plasma norepinephrine and epinephrine concentrations were measured, in the supine resting position and in response to standing, in 10 unselected patients with active acromegaly, in 2 effectively treated acromegalic patients with normal serum growth hormone concentrations and in 15 nonacromegalic normal subjects. Plasma catecholamine concentrations were not significantly related to serum growth hormone levels or to the diastolic blood pressure in the acromegalic patients. Mean (+/- SE) plasma norepinephrine concentrations rose from 211 +/- 28 pg/ml supine to 501 +/- 65 pg/ml after 10 minutes standing in the active acromegalic patients and from 210 +/- 20 pg/ml supine to 502 +/- 54 pg/ml after 10 min standing in the normal subjects. Corresponding plasma epinephrine concentrations were 33 +/- 6 and 60 +/- 14 pg/ml in the acromegalics and 57 +/- 7 pg/ml in the normals. Thus, no abnormality in basal or stimulated plasma catecholamine concentrations was found in acromegalic patients.


Gel filtration profile of circulating immunoreactive thyrotropin and subunits of myxedematous sera. Five sera from hypothyroid patients were passed through gel-filtration columns and the collected fractions were analyzed in three different radioimmunoassays (hTSH-anti-hTSH; hTSHbeta-anti-pTSH; hLH-alpha-anti hLHalpha). For each immunological system, cross reactions were carefully studied. In the hTSH system one main peak was observed at the elution position of the entire molecule, with variable contributions in the subunits area. In the alpha subunit system, every profile showed an asymmetric peak, corresponding roughly to the position of the entire molecule; only two sera exhibited small amounts of free alpha chain. In the beta subunit system, a high molecular weight (100,000 or more) immunoreactive protein was present in every serum. Moreover, cross-reacting intact molecule was eluted as a second peak and in four sera free beta chain was detected. The significance of the big beta subunit, detectable only in the beta assay, is not elucidated.


Studies on the thyroidal defect in an atypical form of Pendred's syndrome. A 52-yr-old woman and her 47-yr-old brother both had large goiters, short stature, nerve deafness, and mild intellectual impairment. Serum levels of thyroid hormones were normal (sister) on borderline low (brother), urinary 127I levels were normal, and perchlorate discharge tests were negative. After adminstration of 125I, we found normal uptakes and retention of istotope by the thyroid, normal serum half-lives for PB 125I, and absence of labelled iodotyrosines in serum or urine. Particulate fractions from both glands had peroxidase activity by in vitro tests for guaiacol oxidation and tyrosine iodination. Samples from different parts of the sister's gland showed a correlation between peroxidase activity in vitro and radioiodine uptake in vivo. We made three separate preparations of thyroglobulin from the sister and one from the brother by gel filtration on 4% agarose. These were similar to each other and to thyrolobulin samples from normal humans when examined by gel electrophoresis both before and after reduction and alkylation. However, the three thyroglobulins from the same gland differed significantly from one another in their composition of most amino acids. This finding indicates that there can be heterogeneity in the protein portion of thyroglobulin, and since the three samples of thyroglobulin were from the same individual, this heterogeneity was probably not of genetic origin. In these patients we could not demonstrate any of the well-established biochemical defects associated with familial goiter. An abnormality in thyroglobulin structure remains in distinct possibility, but we must defer conclusions until there is more known about thyroglobulin heterogeneity and its relationship to hormone biosynthesis.


Inhibitory effect of prednisone on insulin secretion in man: model for duplication of blood glucose concentration. Treatment with oral prednisone (15 mg every 6 h) for 1 day plus a 4-h glucose infusion at 2.8 mg/min kg body weight to 5 normal, healthy individuals raised their blood glucose to 137 +/- 4.5 mg per 100 ml (mean +/- SEM). In order to evaluate the effects of steroid-induced hyperglycemia on insulin responses, a model for the duplication of blood glucose concentration in serial studies was developed. During glucose infusion at 5.7 mg/min kg body weight, the fractional uptake of glucose at the end of infusion (KG) was 2.08 +/- 0.2%/min and the apparent volume of distribution (V) was 285 +/- 10.5 ml/kg. Further increase in the rate of glucose infusion did not affect KG and V. Based on these parameters, KG and V, the stable blood glucose achieved during the prednisone study (C) was duplicated both after short (4 h) and prolonged (28 h) glucose infusions (138 +/- 4.5 and 146 +/- 4.5 mg/100 ml, respectively) at rates calculated as the product of KG.C.V. The effects of prednisone treatment on insulin secretion were examined (1) during fasting, (2) at identical glucose concentrations during glucose infusions at constant rates, and (3) in response to glucose pulse (0.1 g/kg) during the infusions. During fasting, there was a significant elevation of mean blood glucose with prednisone (99 +/- 1.8 mg/100 ml) compared with that in the control study (88 +/- 1.7 mg/100 ml). The plasma IRI, however, remained unchanged (10 +/- 2.3 vs 10 +/- 1.6 muU/ml). During glucose infusions in the presence of similar blood glucose levels, the IRI was lower after prednisone treatment (18 +/- 1.5 muU/ml) than during the short and prolonged glucose infusions (42 +/- 5.1 and 63 +/- 7.0 muU/ml). The insulin response to the glucose pulse also was significantly lower during steroid treatment. Thus, prednisone apparently has an early inhibitory effect on the insulin response to glucose.


Selective hypoaldosteronism despite prolonged pre- and postoperative hyperreninemia in primary aldosteronism. In a prospective study of 7 patients with aldosterone-producing adenoma (APA), long-term (6-72 months) preoperative stimulation of plasma renin activity (PRA) by diuretic therapy (spironolactone plus hydrochlorothiazide) did not prevent selective aldosterone deficiency postoperatively. In all patients aldosterone excretion rate (AER) fell to subnormal values (from a mean of 97 to 2.6 mug/24 h) following removal of APA, although PRA remained elevated. Generalized adrenocortical insufficiency was excluded by the demonstration of normal baseline plasma cortisol and urinary 17-OHCS and the appropriate response to ACTH stimulation. In 6 of 7 patients studied 1-3 months postoperatively, short-term (4 days) sodium deprivation evoked normal increases in PRA, but AER response was blunted (except in 1). Restudy of 3 of 6 patients after 6-12 months revealed that aldosterone production had returned to normal. These results indicate that renin deficiency is not the principal cause of postoperative selective hypoaldosteronism in these patients. On the other hand, they appear to substantiate the possibility raised by in vitro and in vivo studies that spironolactone can directly inhibit aldosterone biosynthesis.


Suppression of plasma renin and plasma aldosterone during water immersion in normal man. Previous studies from this laboratory have demonstrated that the redistribution of blood volume and concomitant relative central hypervolemia induced by water immersion to the neck (NI) results in a significant natriuresis which is quantitative identical to that induced by the acute administration of 2 liters of saline. Since the central hypervolemia induced by NI occurs without concomitant alterations in serum sodium and potassium concentration, the NI model was utilized to assess the role of volume in the regulation of both plasma renin (PRA) and plasma aldosterone (PA) in man. Nine normal subjects were studied on two occasions while in balance on a 10 meq Na, 100 meq K diet; Control and NI. The conditions of seated posture and time of day were identical. Blood for PRA and PA was obtained at 30-min intervals for 6 h. NI produced a profound suppression of PRA as early as 30 min with maximal suppression (62%) by 180 min (P less than 0.001). Recovery from NI was associated with a prompt return to pre-study levels. The changes in PA paralleled those of PRA with regard to both the rapidity and magnitude of the suppression (r = 0.993: P less than 0.001). These data emphasize the importance of central volume per se as a primary determinant of PRA and PA regulation in normal man. Furthermore, the current studies confirm the importance of the renin-angiotensin axis in the control of volume-related changes in PA in normal man. The ability of NI to induce a prompt and parallel suppression of PRA and PA without concomitant alterations in plasma composition, suggests that NI may be a preferred investigative tool for assessing the effects of volume expansion on renin-aldosterone.


Fetal plasma prolactin levels.

Fetal plasma prolactin levels. Prolactin levels were measured by radioimmunoassay in umbilical cord plasma from fetuses, in capillary plasma from neonates, and in venous plasma from adults. The concentrations of prolactin in cord plasma from fetuses having gestational ages of 16 to 19 weeks, 20 to 34 weeks, and 35 to 42 weeks were 53 +/- 16 (mean and SE), 233 +/- 30 and 371 +/- 7 ng/ml, respectively. The prolactin levels decreased to 218 +/- 35 ng/ml during the first neonatal week. The similarity of the patterns of prolactin levels, reported gestational estrogen levels and adrenal weights, as well as the known biological properties of prolactin, estrogen, and ACTH is consistent with the view that these three factors may be involved in the growth of the fetal adrenal cortex and its involution in the newborn.


The secretion rates of deoxycorticosterone and corticosterone in young and elderly men. After intravenous administration of 14C-deoxycorticosterone (DOC) and 3H-corticosterone (B) to 6 young and 7 elderly men, there was no significant difference between the two groups for the total recovery of radioactivity in the urine and neutral extract at the end of 2 days, although the elderly men excreted radioactivity at a significantly slower rate within that time period than young men. Excluding the atypical values for secretion rates (SRs) of DOC and B and for excretion of 17-hydroxycorticosteroids (17-OHCS) for one subject in each age group, the mean SRs for DOC were 0.089 mg/day and 0.047 mg/day, and for B, 3.3 mg/day and 2.0 mg/day, for young and elderly men, respectively. Mean excretion of 17-OHCS for young men was 5.9 mg/day and 4.0 mg/day for elderly subjects. All 3 measures were significantly less for elderly men. For the total sample of 13 men, SRs of DOC correlated significantly with SRs for B and with excretion of 17-OHCS, as did SRs of B with 17-OHCS.


Application of a tissue receptor assay to measurement of serum follitropin (FSH). A rat testes tubule receptor assay, employing 125I-hFSH as the radioligand, has been applied to measurement of human serum follitropin (FSH). Highly purified hFSH LER-1575C was used as the reference preparation. Problems related to interference with uptake of radioligand by receptor, presumably due to non-hormonal serum factors, were minimized by dialysis of the serum prior to TRA. Results obtained by TRA were compared with those obtained by radioimmunoassay (RIA). The mean index of discrimination (TRA/RIA) for a series of 11 postmenopausal sera (PM) was 5.4 and the mean index of precision was 0.063. Sensitivity of the TRA was 9 ng hFSH/ml. Postmenopausal serum averaged 81 ng/ml, PM on estrogen therapy averaged less than 9 ng/ml, Turners Syndrome was 40 ng/ml. No FSH was detected in hypophysectomized serum, in normal male serum or in blood bank serum. PM levels by TRA were similar to those obtained by others with RIA after multiplying by the conversion factor of 5.4.


Immunoreactive somatomedin B in urine.

Immunoreactive somatomedin B in urine. Urinary excretion by 8 normal adult subjects of immunoreactive somatomedin B was 27.6 +/- 4.4 mug between 1000 h and 1400 h compared to a mean plasma concentration at 1200 h of 5.9 +/- 0.9 mug/ml. Free somatomedin B in urine averaged 85.9%, although in the plasma of the same subjects all but less than 5% was bound to serum proteins.


Adipose tissue cellularity and lipolysis. Response to exercise and cortisol treatment. Male rats a 5 wk of age were subjected to 13 wk of intensive treadmill running to study the effect of exercise on adipose tissue cellularity and lipolysis. Untrained controls of the same age remained sedentary in their cages for the duration of the experiment. Adipocyte numbers were similar in eqidiymal fat pads from trained and untrained rats (12.7 plus or minus 1.3 X 10(6) vs. 15.3 plus or minus 1.3 X 10(6) cells/pad), however trained rats had smaller fat pads containing smaller cells (0.09 plus of minus 0.01 vs. 0.20 plus or minus 0.04 mug triglyceride/cell). Adipocytes from trained rats possessed greater epinephrine-sensitive lipase activity than sedentary rats on a per cell, per milligram protein, per gram adipose tissue, or per fat pad basis. Although the smaller cells of the trained rats had greater epinephrine-sensitive lipase activity than the larger cells of the untrained rats, lipolysis was positively correlated with cell size within both treatment groups. Cortisol treatment of intact animals did not significantly affect in vitro adipose tissue lipolysis. The results of this study indicate that exercise training increased the potential of adipose tissue cells to release free fatty acids in response to epinephrine stimulation. Exercise training initiated at 5 wk of age had only a small effect on adipose tissue cell numbers but significantly decreased cell size.


Experimental renal osteodystrophy. The response to 25-hydroxycholecalciferol and dicholomethylene diphosphate therapy. Bone mineral and matrix maturation in chronically uremic, nonacidotic rats were investigated after 25-hydroxcholecalciferol (25OHD) and/or dichloromethylene diphosphonate (C12MDP) therapy utilizing bromoform-toluene density gradient fractionation and X-ray diffraction analyses. The bromoform-toluene density gradient analyses demonstrated that the progressive accumulation of less dense, more immature bone characteristic of progressive uremia was reversed by 25OHD and/or C12MDP therapy for a 2-wk period, and that after 4 wk of therapy the maturational profile of bones from chronically uremic animals treated with 250HD and/or C12MDP was comparable to that from nonuremic littermates. X-ray diffraction analysis revealed that by the 4th wk of therapy with 25OHD and C12MDP both the degree of crystallinity and the crystal size/perfection parameters in the uremic bones were comparable to those of nonuremic, pair-fed control littermates. Treatment for 4 wk with 25OHD resulted in enlarged and/or more perfect apatite crystallites, while C12MDP alone slightly inhibited crystal growth and/or perfection after 2 wk of treatment. Soft tissue calcification was diminished in uremic animals treated for 4 wk with C12MDP or a combined C2MDP/25OHD regimen, the latter being much more effective in this regard. The accumulated data in this study support the premise that the attendant accelerated bone resorption, soft tissue calcification, and abnormal mineralization and maturation of the skeletal tissue, well documented to characterize experimental ranal insufficiency, may be alleviated with therapeutic dosages of 25OHD and/or C12MDP.


The electrophysiological effects of ouabain on sinus node and atrium in man. Electrophysiological studies were performed in 16 patients before and 30 min after intravenous administration of ouabain (0.1 mg/kg). P-A interval (mean+/-SEM) was 40+/-2.1 ms before and 44+/- 1.5 ms after ouabain (P less than 0.001). Atrial effective and functional refractory periods (ERP and FRP) were measured in all patients during sinus rhythm and during driving at equivalent paced rates in 12 patients. The mean atrial ERP and FRP during sinus rhythm were, respectively, 244+/-10.5 and 307+/-11.0 ms before and 253+/-9.7 and 318+/-11.4 ms after infusion of ouabain (NS). Mean atrial ERP and FRP during driving were, respectively, 231+/-15.3 and 264+/-14.9 ms before and 266+/-18.6 and 296+/-19.7 ms after ouabain (P less than 0.01 and P less than 0.01). Mean sinus cycle length and sinus recovery times were, respectively, 887+/-31.2 and 1,113+/-38.7 ms before and 905+/-38.2 and 1,008+/-30.7 ms after infusion of ouabain (NS and P less than 0.005). Calculated sinoatrial conduction times before and after ouabain were 90+/-6.8 and 110+/-8.5 ms, respectively (P less than 0.005). In summary, ouabain produced depression of intraatrial conduction as manifested by increase in P-A interval and atrial effective and functional refractory periods. Ouabain significantly increased calculated sinoatrial conduction time without significant effect on spontaneous sinus cycle length.


The metabolism of 3alpha, 7alpha, 12alpha-trihydorxy-5beta-cholestan-26-oic acid in two siblings with cholestasis due to intrahepatic bile duct anomalies. An apparent inborn error of cholic acid synthesis. Studies were carried out in a family in which two children with cholestasis due to intrahepatic bile duct anomalies were shown to have increased amounts of the cholic acid precursor, 3alpha, 7alpha, 12alpha-trihydorxy-5beta-cholestan-26-oic acid (THCA). The metabolism of THCA was studied in one of these patients after an intravenous injection of (3H)THCA, and the cause of the increased amounts of THCA in this condition was found to be due to a metabolic defect in the conversion of this compound into cholic acid. A small amount of (3H)cholic acid was also identified after (3H)THCA administration, confirming that this metabolic defect was incomplete. Varanic acid (3alpha, 7alpha, 12alpha, 24xi-tetrahydorxy-5beta-cholestan-26-oic acid), a metabolite of THCA, could not be identified in either of these patients. By assuming that this compound would be conjugated and excreted if the metabolic block occurred after the formation of varanic acid, the defect in these patients appears to be due to a deficiency of a 24-hydroxylating enzyme system required to convert THCA into varanic acid. This condition appears to be transmitted in an autosomal recessive fashion, because the two affected patients were of opposite sex, and neither a normal sibling nor the two parents have increased amount of THCA in their bile.


Enzymatic degradation of uric acid by uricase-loaded human erythrocytes. Erythrocytes containing pig liver uricase have been prepared by hypotonic hemolysis in the presence of the enzyme. Uricase is shown to be active within the erythrocytes and to degrade uric acid as rapidly as it enters the cells when high intracellular enzyme concentrations are employed. The kinetics and characteristics of uric acid entry are shown to be the same for hemolysed and normal erythrocytes. At physiological concentrations of uric acid, loaded erythrocytes can degrade a maximum of about 21 mumol uric acid/liter erythrocytes per min. The possible application of enzyme-loaded erythrocytes to medicine is discussed.


The mechanism of the effect of aspirin on human platelets. I. Acetylation of a particulate fraction protein. Aspirin (acetylsalicylic acid) inhibits platelet prostaglandin synthesis and the ADP- and collagen-induced platelet release reaction. The mechanism of the inhibitory effect is unknown but may involve protein acetylation, since aspirin acetylates a variety of substrates, including platelet protein. We have examined the relationship between protein acetylation and aspirin's physiologic effect on platelets. Suspensions of washed human platelets were incubated at 37 degrees C with (3H)aspirin, and incorporation of radioactivity into protein was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Exposure to (acetyl-3H)aspirin but not (aromatic ring-3H)aspirin resulted in radioactive labeling of three platelet proteins, suggesting that the drug acetylates these three proteins. The acetylation of two of the proteins (located in the supernatant fraction) was not saturable, implying that these reactions may not be physiologically significant. Acetylation of the third protein, approximate mol wt 85,000 (located in the particulate fraction), saturated at an aspirin concentration of 30 muM and was complete within 20 min. Platelets prepared from aspirin-treated donors did not incorporate any (acetyl-3H)aspirin radioactivity into the particulate protein for 2 days after drug treatment and did not show full pretreatment uptake of radioactivity for 12 days thereafter. The course of increasing incorporation of (acetyl-3H)aspirin radioactivity parralleled that of platelet turnover. Therefore, in addition to its saturability, acetylation of the particulate fraction protein by aspirin was permanent. In two respects, the inhibition of platelet function by aspirin correlates well with the aspirin-mediated acetylation of the particulate fraction protein. Both persist for the life-span of the aspirin-treated platelet, and both occur at a similar saturating aspirin concentration. The evidence suggests that the physiologic effect of aspirin on human platelets is produced by acetylation of a single protein located in the particulate fraction. The acetylated protein may be related to cyclo-oxygenase, the prostaglandin G2 biosynthetic enzyme.


Thyrotropin-induced hyperthyroidism caused by selective pituitary resistance to thyroid hormone. A new syndrome of "inappropriate secretion of TSH". An 18-yr-old woman with clinical and laboratory features of hyperthyroidism had persistently elevated serum levels of immunoreative thyrotropin (TSH). During 11 yr of follow-up there had been no evidence of a pituitary tumor. After thyrotropin-releasing hormone (TRH), there was a marked increase in TSH and secondarily in triiodothyronine (T3), the latter observation confirming the biologic activity of the TSH. Exogenous T3 raised serum T3 and several measurements of peripheral thyroid hormone effect, while decreasing serum TSH, thyroxine (T4), and thyroidal radioiodine uptake. After T3, the TRH-stimulated TSH response was decreased but was still inappropriate for the elevated serum T3 levels. Dexamethasone reduced serum TSH but did not inhibit TRH stimulation of TSH. Propylthiouracil reduced serum T4 and T3 and raised TSH. This patient represents a new syndrome of TSH-induced hyperthyroidism, differing from previous reports in the absence of an obvious pituitary tumor and in the responsiveness of the TSH to TRH stimulation and thyroid hormone suppression. This syndrome appears to be caused by a selective, partial resistance of the pituitary to the action of thyroid hormone. This case is also compared with previous reports in the literature of patients with elevated serum levels of immunoreactive TSH in the presence of elevated total and free thyroid hormones. A classification of these cases, termed "inappropriate secretion of TSH," is proposed.


Reduced peripheral conversion of thyroxine to triiodothyronine in patients with hepatic cirrhosis. The role of liver in the peripheral conversion of thyroxine (T4) to triiodothyronine (T3) was studied in normal subjects and patients with alcoholic liver disease by measurement of thyrotrophin (TSH) and total and free T4 and T3 in randomand serial serum samples. Also, T4 to T3 conversion rates and T3 disposal rates were compared by noncompartmental analysis. While the mean total serum T4 values were similar for the two groups, 8.6 and 8.1 mug/kl, the mean free T4 value was significantly higher in the cirrhotic patients (3.3 ng/dl) than in the normal subjects (2.1 ng/dl, P less than 0.001). The mean serum T3 value, 85 ng/dl, was significantly reduced in the hepatic patients as compared to a mean serum T3 value of 126 ng/dl in the normal subjects (P less than 0.001), while the free T3 value was 0.28 ng/dl in both groups. The reduction of the serum total and free T3 values were closely correlated with the degree of liver damage, as indicated by elevation of serum bilirubin (r equal -0.547) and reduction of serum albumin (r equal 0.471). The mean serum TSH level was 3.1 muU/ml in the normals and 7.1 muU/ml in the cirrhotic aptients ( less than 0.001). 15% of the hepatic patients had serum TSH values above 10 muU/ml, which, however, did not correlate with any of the four liver function tests studied. Serial blood sampling from two convalescing patients with alcoholic hepatitis showed a gradual normalization of serum TSH and T3 levels as the liver function improved. After oral T4 administration, 0.25 mg/day for 10 days, three of four cirrhotic patients studied failed to raise their serum T3 values. The mean T4 to T3 conversion rate of seven normal subjects was 35.7%. The mean T4 to T3 conversion rate of four cirrhotic patients studied was significantly reduced to 15.6% (P less than 0.001). The mean disposal rates of T4 and T3 of the normal subjects were 114 and 34 mug/day, respectively. The ratio of T4 disposal to T3 disposal was 3.5. In contrast, the mean T4 disposal rate, 82 mug/day, and the mean T3 disposal rate, 10 mug/day, were both reduced in the cirrhotic patients. Their ratio of T4 disposal to T3 disposal was 7.9. These findings suggest that impairment of T4 conversion in patients with advanced hepatic cirrhosis may lead to reduced T3 production and lowered serum T3 level. Therefore, the liver is one of the major sites of T4 conversion to T3.


The role of iron in the pathogenesis of porphyria cutanea tarda. II. Inhibition of uroporphyrinogen decarboxylase. Porphria cutanea tarda is characterized biochemically by excessive hepatic synthesis and urinary excretion of uroporphyrin I and 7-carboxylporphyrins. This pattern of excretion suggest an impaired ability to decarboxylate uroporphyrinogen to the paired ability to decarboxylate uroporphyringen to the 4-carboxyl porphyrinogen, coproporphyrinogen, a reaction catalyzed by the enzyme uroporphyringen decarboxylase. Because clinical evidence has implicated iron in the pathogenesis of porphyria cutanea tarda, these experiments were designed to study the effect of iron on uroporphyrinogen decarboxylase in procine crude liver extracts. Mitochondria-free crude liver extracts were preincubated with ferrous ion and aliquots were assayed for uroporphyrinogen decarboxylase activity. Uroporphyrinogens I and III, the substrates for the decarboxylase assay, were prepared enzymatically from (3H)porphobilinogen. The products of the decarboxylase reaction were identified and quantitated by three methods: (a) extraction into 1.5 N HCl and spectrophotometric quantitation; (b) adsorption onto talc, esterification, paper chromatographic identification, and quantitation by liquid scintillation counting; and (c) adsorption onto talc, esterification, thin-layer chromatographic identification on silica gel, and quantitation by liquid scintillation counting. The thin-layer scinllation method proved most sensitive as it was the only method which accurately identified and quantitated the 7-carboxyl porphyrin reaction product. Uroporphyrinogens I and III were decarboxylated at the same rate by porcine hepatic uroporphyrinogen decarboxylase, and the addition of iron induced marked inhibition of the decarboxylase activity. Ortholpehanthroline blocked the inhibitory effect of iron. The inhibition of uroporphyrinogen decarboxylase by ferrous ion, coupled with its previously reported inhibitory effect on uroporphyrinogen III cosynthetase, provides a possible biochemical explanation for the pattern of urinary porphyrin excretion observed in patients with porphyria cutanea tarda and the clinical association with disordered iron metabolism.


Decrease in serum immunoreactive parathyroid hormone in rats and in parathyroid hormone secretion in vitro by 1,25-dihydroxycholecalciferol. The present study determined the effects of 1,25-dihydroxycholecalciferol on serum immunoactive parathyroid hormone and on parathyroid hormone secretion in vitro. Rats injected i.p. with 1,25-dihydroxycholecalciferol, 130 pmol (2 U)/140 g body wt, which is probably a physiologic dose, had a significant 43% decrease in serum immunoreactive parathyroid hormone at 4 h. In addition, this dose of 1,25-dihydroxycholecalciferol inhibited the serum immunoreactive parathyroid hormone response to hypocalcemia induced by phosphate injection. Because the increment in serum immunoreactive parathyroid hormone was less but the decrement in serum calcium more in phosphate plus 1,25-dihydroxycholecalciferol-treated than in phosphate plus vehicle-treated rats, the impaired serum immunoreactive parathyroid hormone response to 1,25-dihydroxycholecalciferol could not be attributed to the change in serum calcium. In studies of parathyroid hormone secretion from bovine parathyroid tissue in vitro, the concentration of 1,25-dihydroxycholecalciferol used for most experiments was 1nM, which is in the range found in rat serum. 1,25-Dihydroxycholecalciferol at 1 or 100 nM significantly inhibited parathyroid hormone secretion when medium calcium concentration was normal (1.5 mM), high (3.0 mM), and low (1.0 mM). Maximum inhibition ranged from 19 to 74%; inhibition was generally seen after 2 h of incubation; and inhibition was sustained or progressive thereafter. Vitamin A, 0.1 muM, caused a marked stimulation of parathyroid hormone secretion. 1,25-Dihydroxycholecalciferol at 1 nM markedly reduced (44%) the effect of vitamin A to stimulate parathyroid hormone secretion. This effect of 1,25-dihydroxycholecalciferol was maximal at 1 h and persisted thereafter. Another steroid, hydrocortisone, 10 muM, did not inhibit parathyroid hormone secretion, suggesting that the 1,25-dihydroxycholecalciferol effect was not a nonspecific inhibitory effect on parathyroid cells. Because other workers have shown that parathyroid hormone directly stimulates 1,25-dihydroxycholecalciferol secretion, our results are consistent with the concept that there is a feedback loop where parathyroid hormone directly stimulates secretion of 1,25-dihydroxycholecalciferol, which in turn directly inhibits secretion of parathyroid hormone.


The mechanism of action of a single dose of methylprednisolone on acute inflammation in vivo. A model system for the study of inflammation in vivo has been developed using the 16-h polyvinyl sponge implant in the rat. This system allows for simultaneous measurement of in vivo chemotaxis, volume of fluid influx, and fluid concentrations of lysosomal and lactic dehydrogenase (LDH) enzymes. In addition, the enzyme content of inflammatory fluid neutrophils may also be determined. A parallel time course of neutrophil and lysosomal enzyme influx into sponge implants was observed. This was characterized by an initial lag phase and a rapid increase between 5 and 16 h. The origin of supernatant LDH and lysosomal enzymes was studied with anti-neutrophil serum to produce agranulocytic rats. Inflammatory fluid in these rats was almost acellular and contained decreased concentrations of beta glucuronidase (-96%) and LDH (-74%). In control rats all of the supernatant beta glucuronidase could be accounted for by cell death and lysis, as estimated from measurements of soluble DNA. Only 15-20% of the LDH activity could be accounted for on the basis of cell lysis. The remainder was derived from neutrophil-mediated injury to connective tissue cells. Large intravascular doses of methylprednisolone markedly inhibited neutrophil influx into sponges and adjacent connective tissue. Secondary to decreased neutrophil influx, fewer neutrophils were available for lysis, and lysosomal enzyme levels in inflammatory fluid decreased. No evidence for intracellular or extracellular stabilization of neutrophil lysosomal granules by methylprenisolone was found.


Suppression of sleep-related prolactin secretion and enhancement of sleep-related growth hormone secretion. Methysergide, a clinically-used blocker of serotonin receptors, was administered to 10 normal young men at a dose of 2 mg every 6 h for 48 h. After drug treatment, serum levels of growth hormone during sleep were 41.9% higher than placebo values (less than 0.001). In contrast, drug treatment was associated with a 36.4% decrease in stimulated growth hormone secretion during insulin tolerance testing  (P less than 0.01). These opposite effects of methysergide suggest that different mechanisms are responsible for sleep-related and insulin-induced growth hormone secretion. Accordingly, data obtained with pharmacologic stimuli may lead to erroneous inferences regarding physiologic growth hormone control mechanisms. Administration of methysergide profoundly suppressed sleep-related prolactin secretion; overall nocturnal mean prolactin fell by 70.3% from 4.30+/-0.19 to 1.28+/-0.06 ng/ml (P less than 0.0001). It appears that serotonin may be significant modulating neurotransmitter for the control of growth hormone secretion, limiting sleep-related release, and enhancing insulin-induced release. It seems likely from these data that the role of serotonin in the control of prolactin secretion is relatively more important, since serotonin receptor blockade dramatically reduced sleep-related prolactin secretion.


Subunit structure of factor VIII antigen synthesized by cultured human endothelial cells. Cultured human endothelial cells were labeled with (3H)leucine, and the radioactive Factor VIII antigen present in the postculture medium was isolated by double anitbody immunoprecipitation and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reduction with dithiothreitol. The Factor VIII antigen synthesized by cultured endothelial cells was found to contain the same single polypeptide subunit (mol wt 225,000) present in plasma Factor VIII antigen. These results suggest that in vivo, the endothelial cell is a major site of synthesis of circulating Factor VIII antigen.


Complete absence of the third component of complement in man. A 4-yr-old female patient who has recurrent infections with encapsulated bacteria and gramnegative organisms was found to have a complete absence of total hemolytic complement and C3. Total hemolytic complement was reconstituted by the addition of functionally pure C3. With the exception of a moderately reduced homolytic C4, all other C components, measured homolytically and by radial immunodiffusion, were present in normal amounts. By Ouchterlong analysis, the patient's serum contained C3b inactivator and properdin but no antigenic C3. Activation of the alternate pathway was examined by purified cobra venom factor (CVF) and inulin. Neither of these substances led to activation of properdin factor B to B. On addition of partially purified Cordis C3, in four out of four instances and with different preparations of Cordis C3, activation of factor B to B occurred in the inulin-serum-C3 mixture. In contrast, activation of factor B to B occurred only once out of four times with CVF-serum-C3 mixtures. Immune adherence was found to be normal in the patient's serum and could be removed by anti-C4 antiserum of hydrazine treatment. A marked opsonic defect was present against Escherichia coli. Serum bactericidal activity against a rough strain of E. coli was also defective. The ability to mobilize an infalmmatory response was examined by Rebuck skin window technique. A delay in neutrophil migration occurred until the 6th h. In vitro lymphocyte transformation and serum immunoglobulins were normal. The proportion of peripheral blood T cells forming spontaneous sheep erythrocyte rosettes and the percentage of B cells forming EAC rosettes by the C3 receptor were normal. The significance of the absence of C3 in our patient is emphasized by the increased number of infections with encapsulated bacteria and the decreased functional biological activities of the C system, important in host defense mechanism(s).


Metabolism of properdin in normal subjects and patients with renal disease. Properdin deposition has been recognized in glomeruli of patients with acute and chronic nephritis and lupus nephritis, and low serum properdin levels have been found in these disorders. These findings suggest that properdin may be involved in the production of glomerular damage and that low properdin levels may be due to hypercatabolism. The study was designed to examine the metabolism of properdin in normal subjects and to look for an abnormality in five patients with systemic lupus erythematosus with renal involvement and in six patients with membranoproliferative glomerulonephritis or dense deposit disease (MPGN). Highly purified human properdin was prepared by elution from zymosan, followed by DEAE-cellulose and carboxymethyl-Sephadex chromatography, and labeled with 125I by the iodine monochloride method. Parameters of metabolism were determined by monitoring plasma and urinary radioactivity at frequent intervals after the intravenous injection of 1-2 muCi of labeled material. The fractional catabolic rate (FCR) of properdin in normal subjects was found to have a very narrow range of 0.78-1.0,% of the plasma pool per hour (mean 0.95%). In systemic lupus erythematosus, the FCR was regularly elevated with a range of 1.21-2.30% (mean 1.70%). In MPGN, FCR was elevated in three patients (1.22, 1.94, and 2.08%) and within or below the normal range in three (0.78, 1.00, and 1.00%). Properdin levels were reduced in two patients who had the highest FCR's noted in the study. Properdin synthetic rates in normals varied from 4.1 to 14.3 mug/kg per h (mean 9.1) and was not found to be reduced in any patient. Properdin catabolism was found to be normal in a patient deficient in the C3b inactivator. These studies show that properdin is hypercatabolized in patients with renal disease and that decreased properdin levels when they occur in these patients can be entirely explained on the basis of this hypercatabolism.


Differences between purified ectopic and normal alpha subnits of human glycoprotein hormones. "Ectopic" proteins, not distinguished immunologically from the common alpha subunit of the human glycoprotein hormones, were purified approximately 10,000-fold from a gastric carcinoid tumor (A.L.-alpha) and from tissue culture medium of bronchogenic carcinoma cell lines (ChaGo-alpha). The purified A.L.-alpha was homogeneous by sodium dodecyl sulfate (SDS) gel electrophoresis while the purified ChaGo-alpha showed multiple components, some of which represented aggregated species. In SDS gel electrophoresis, the apparent molecular weights of A.L.-alpha (15,000) and dithioerythritol-reduced ChaGo-alpha (13,000) were significantly lower than those of the alpha subunits of human chorionic gonadotropin (hCG-alpha), luteinizing hormone, follicle-stimulating hormone, or thyroid-stimulating hormone (22,000-23,000). Binding experiments with [35S]-SDS suggested that these apparent differences in molecular weight resulted, at least in part, from diminished binding of the SDS by the normal compared to the ectopic alpha subunits. In gel chromatography, the apparent molecular weights of A.L.-alpha (27,000) and ChaGo-alpha (30,000) were slightly higher than those of normal alpha subunits (23,000-24,000). Both A.L.-alpha and ChaGo-alpha were not distinguished from hCG-alpha in ion-exchange chromatography. The composition of A.L.-alpha was similar to that of hCG-alpha in 13 amino acids but showed decreased phenylalanine and increased valine; glucosamine was identified in both A.L.-alpha and hCG-alpha. Under conditions in which hCG-alpha combined with the hCG beta subunit (hCG-beta) to produce 95% of the expected gonadotropin-binding activity in a rat testis radioreceptor-assay, A.L.-alpha incubation with hCG-beta resulted in only 2% of the expected activity, and ChaGo-alpha incubation with hCG-beta produced no detectable activity. These characteristics of ectopic alpha subunits may reflect abnormalities of neoplastic protein synthesis or carbohydrate attachment which result in polypeptides with chemical and immunologic similarity to normal subunits but with differences in physical and combining properties; alternatively, the ectopic subunits may represent as yet unrecognized alpha precursor forms.


Isolation and properties of the mixed lipid micelles present in intestinal content during fat digestion in man. To evaluate better the physicochemical characteristics of human fat digestion, a method was developed which allowed characterization of the bile acid-lipid mixed micelles of the aqueous phase of post-prandial duodenal fluid. Duodenal fluid was collected after a 36-g fat breakfast for two 90-min periods and for 60 min after i.v. cholecystokinin and was ultracentrifuged at 15,400,000 g-min. The aqueous phase was isolated, passed through a 200-nm filter, and the mixed micelles were concentrated by an ultrafiltration procedure using a 1.5-nm filter. The 1.5-nm retentate was eluted from Sepharose 6B columns with 1.5-nm filtrate for both preequilibration fluid and eluent. 1.5-nm filtrate approximated the monomer concentrations. Each sample was assayed for bile acid, fatty acid, lecithin, lysolecithin, protein, cholesterol, and counterions (pH, Na+, K+, Ca2+). Constituents were concentrated only on the 1.5-nm filter. On gel permeation chromatography, coincident peaks were observed for bile acid, fatty acid, lysolecithin, and cholesterol; and were eluted with a Kav range of 0.50-0.68 (corresponding to a Stokes radius of 2.3-3.5 nm). An average density of 1.25 and coincident peaks of bile acid and fatty acid were found for the mixed micelles on sucrose density gradients. The regression lines of micellar fatty acid, lysolecithin, and cholesterol vs. bile acid gave a stoichiometry of 1.4 mol fatty acid, 0.15 mol lysolecithin, and 0.06 mol cholesterol for each mole of bile acid. Mixed micelles were homogeneous in composition. These results provide direct evidence for the existence of the postprandial mixed micelle and describe several of its physicochemical properties.


25-hydroxycholecalciferol-enhanced bone maturation in the parathyroprivic state. In vitro evidence presently favors a direct osteolytic effect of biologically active vitamin D metabolites. Studies were designed to evaluate the effect of 25-hydroxycholecalciferol (25OHD3) on bone collagen and mineral maturation in vivo and its dependence on parathyroid hormone (PTH). After treatment of sham-operated control and parathyroidectomized (PTX) mature rats with either 25OHD3 or an oil vehicle for 2 wk, tibial bone mineral-collagen maturation was quantitated by bromoform-toluene density gradient fractionation techniques. Intestinal calcium absorption was measured by in vivo 45Ca transport procedures. In contrast to the control group, the response to 25OHD3 of PTX rats was dramatic. Bone mineral and matrix maturation were both accelerated by 25OHD3 treatment without concomitant reduction in total bone mineral or collagen content or changes in the intestinal calcium absorption. These observations support the premise that biologically active vitamin D metabolites stimulate bone tissue maturation, and that PTH is not required in this regard.


Comparison of agents producing a neutrophilic leukocytosis in man. Hydrocortisone, prednisone, endotoxin, and etiocholanolone. To study the potential application of glucocorticosteroid administration for the measurement of the bone marrow neutrophil reserve response, blood neutrophil count changes were measured in normal subjects after the administration of intravenous hydrocortisone (25, 50, 100, 200, and 400 mg) and oral prednisone (5, 10, 20, 40, and 80 mg). The upper three doses of both steroids increased the blood neutrophil count by approximately 4,000 cells/mm3. The neutrophilia occurring after hydrocortisone (200 mg) and/or prednisone (40 mg) was compared with that observed after endotoxin (0.8 ng/kg) and etiocholanolone (0.1 mg/kg) in 14 normal subjects, 7 patients with Wegener's granulomatosis on cyclophosphamide therapy and 10 patients with chronic idiopathic neutropenia. The normal responses (mean increase of blood neutrophils/mm3 above base line +/- 1 SEM) were: hydrocortisone 4,220 +/- 320, prednisone 4,610 +/- 360, endotoxin 6,060 +/- 880, and etiocholanolone 3,780 +/- 440. In the patient studies, etiocholanolone gave the smallest mean responses, but, in general, the results were similar for all agents. These data indicate that these glucocorticosteroids can be used as equivalent agents to endotoxin and etiocholanolone for measuring the neutrophil reserve response.


Beneficial effects of penicillamine treatment on hereditary avian muscular dystrophy. Hereditary muscular dystrophy in chickens of the New Hampshire strain was treated with penicillamine from the 9th day after hatching to the 425th day. The adult maintenance dose for males was 50 mg/kg per day and for females, 13-65 mg/kg per day. In avian dystrophy, deterioration of the muscle fibers is evidenced in the 2nd mo by an inability of the birds to rise after falling on their backs and by a progressive rigidity of the wings. The drug delayed the onset of symptoms and partially alleviated the debilitating aspects of the disease. Penicillamine produced three major improvements: (a) better righting ability when birds were placed on their backs; (b) greater wing flexibility; (c) and suppression of plasma creatine phosphokinase activity. The results are statistically analyzed and discussed in relationship to Duchenne dystrophy. Normal birds were not affected by penicillamine as judged by these parameters. The rationale for using penicillamine, a sulfhydryl compound with reducing properties, was (a) to attempt to protect essential thiol enzymes in the anabolic and glycolytic pathways against inactivation and (b) to prevent collagen cross-linking and deposition in muscle. Although the precise mechanism of drug action has not been determined. the possible role of penicillamine in mitigating the symptoms of genetic dystrophy in man is under consideration. Further, penicillamine may have a more generalized application i the prevention of contractures in a variety of neuromuscular disorders.


The pathogenesis of post-obstructive diuresis. The role of circulating natriuretic and diuretic factors, including urea. To investigate the pathogenesis of post-obstructive diuresis, a state of functional "anuria" during ureteral obstruction was created in awake rats by (a) bilateral obstruction (BO); (b) unilateral obstruction and contralateral nephrectomy (UO-Nx); or (c) unilateral obstruction and continuous i.v. reinfusion of urine from the intact contralateral kidney (UO-reinf). These groups were compared with unilaterally obstructed (UO) and sham-operated control (sham) rats. After release of obstruction of 24 h duration, mean urine flows (V) and sodium excretion rates (UNaV) were significantly elevated above those of sham rats in BO, UO-Nx, and UO-reinf animals, but slightly decreased in UO rats. Glomerular filtration rates were comparably depressed in UO, BO, UO-Nx, and UO-reinf rats. These results suggest that post-obstructive diuresis is due to one or more circulating diuretic factors that are normally excreted in the urine, and which, when retained )as in BO or UO-Nx rats) or returned to the circulation (as in UO-reinf rats), exert a diuretic affect. In additional experiments, UO rats infused with urea exhibited post-obstructive diuresis, if extracellular volume contraction was prevented. This result suggests that urea may be an important diuretic factor in post-obstructive diuresis, but does not exclude possible roles for other humoral factors. The intact kidney of UO-reinf rats displayed a massive unilateral diuresis and natriuresis, further suggesting the presence of potent diuretic factors in the urine. A marked increase in the fractional excretion of glomerular filtrate (V/GFR) by the intact kidney suggests that this diuresis may be attributable, in part, to impaired proximal reabsorption.


Time course of and stimuli to compensatory growth of the lung after pneumonectomy. Left pneumonectomy in the mature rat led to an increase of [3Ha1 thymidine incorporation into DNA of the remaining lung in the first 3 postoperative days, and resulted in a subsequent 38% increase of lung weight and 41% increase of lung tissue volume measured 1 wk after surgery. Despite these early changes, total lung volume (TLV) did not increase until the 2nd postoperative wk, reaching values 33% greater than in controls. Analysis of lung pressure-volume curves revealed that lung recoil was increased at low lung volumes 1 wk after surgery, but returned to normal by the 2nd postoperative wk, suggesting that synthesis of both lung elastin and collagen had occurred by this time. Increased inspired oxygen concentration (28% or 35%) during the 1st but not the 2nd postoperative wk abolished the change in TLV without influencing the increase in lung weight, while diminished inspired oxygen (17% or 14%) accentuated the postoperative increase in TLV. Lung pressure-volume curves demonstrated changes in distensibility at low lung volumes, suggesting that oxygen may have influenced synthesis or cross-linking of lung elastin. Alterations of minute ventilation in the postoperative period produced by 3% CO2 did not influence the compensatory growth process, nor did administration of cyclophosphamide. These studies suggest that postpneumonectomy lung growth is a two-phase process, beginning with cell proliferation and increased tissue volume, followed by increasing lung volume associated with formation of lung structural proteins. The latter process is profoundly influenced by inspired oxygen concentration in the early postoperative period.


Sex steroid influence on triglyceride metabolism. Triglyceride metabolism was investigated in groups of fed and fasted rats after 21 days of parenteral estradiol (5 mug daily), progesterone (5 mg daily), or the two steroids in combination. Results were compared with control groups receiving an oil solvent alone. In rats given estradiol separately or combined with progesterone, hypertriglyceridemia was uniformly associated with increased plasma triglyceride entry, estimated with the i.v. Triton WR1339 technique. Progesterone alone had no effect on these parameters. Plasma postheparin lipolytic activity (PHLA), adipose, mammary gland, and protamine-resistant liporotein lipases (LPL) were significantly increased in progesterone-treated rats and significantly decreased in rats receiving estradiol with the exception of mammary gland LPL, which was also increased to a slight extent. The combined regimen reduced plasma PHLA and increased protamine-resistant adipose, and mammary gland LPL activity. Sex steroid treatments had minimal effects on plasma glucose and free fatty acid concentrations, but all increased plasma insulin significantly. Hyperinsulinemia did not parallel changes in body weight or other measured parameters. Linear regression analyses revealed that plasma triglyceride concentrations in all fed, treated rats correlated significantly with triglyceride entry but not very uniformly with plasma or tissue LPL activity. We conclude that estradiol, unlike progesterone, has substantial lipemic effects in the rat which relate best to triglyceride entry. Hyperinsulinemia, changes in body weight, plasma PHLA, and tissue LPL activities did not consistently predict the influence of sex steroid treatment on plasma triglyceride concentrations.


Effect of collateral flow on epicardial and endocardial lysosomal hydrolases in acute myocardial ischemia. Early changes in lysosomal enzymes must occur if their role is significant in irreversible myocardial injury. Therefore, we ligated the anterior descending coronary artery in 14 dogs and after 60 min excised epicardial and endocardial samples from the ischemic and adjacent normal heart. The collateral flow measured with radioactive microspheres in the endocardial samples averaged 19% of control. The muscle was disrupted and fractionated by ultracentrifugation into nuclear pellet (NP), heavy lysosomal pellet (HL), light lysosomal pellet (LL), microsomal pellet (M) and supernate (S). Electron microscopy demonstrated changes characteristic of sichemia in whole tissues and sedimented fractions. Acid phosphatase reaction product was present in residual bodies in the HL fraction and membrane-bound vesicles in the LL fraction and in the intact tissue. Significant decreases in the specific activity of N-acetyl-beta-glucosaminidase and beta-glucuronidase occurred in the endocardial LL fraction, while significant increases in both were found in the ts fraction (P less than 0.05). Losses of acid phosphatase occurred in both LL and S fractions. Moreover, decreases of total N-acetyl-beta-glucosaminidase in the HL fraction and of total beta-glucuronidase and acid phosphatase in the LL fraction were positively correlated (P less than 0.01) with the degree of ischemia measured with radioactive microspheres. Only insignificant enzymatic changes were found when the collateral flow was greater than 40%, and the differences were less significant in epicardial samples where the flow averaged 29%. The early loss of enzymes from the lysosomal fractions in severe ischemia suggests a role for lysosomal hydrolases in the necrosis that follows coronary occlusion.


The perturbation of thrombin binding to human platelets by anions. Thrombin binds with high affinity to specific cell-surface receptors on washed human platelets. We present experiments indicating that thrombin binding correlates withe the release reaction when binding is perturbed by anions. Marked differences in the affinity of human 125I-thrombin for platelets wer observed in various isotonic buffers at pH 7.4. At low concentrations of thrombin (0.001-0.01 U/ml), binding was 5-fold greater in Tris-sodium acetate and 12-fold greater in Tris-sodium cacodylate than in Tris-sodium chloride. These anion-induced changes in 125I-thrombin binding paralleled changes in [14C] serotonin release when both parameters were measured in the same platelets. Thus, equivalent release occurred for equal amounts of thrombin bound in all buffers, even though the thrombin concentration varied by up to 30-fold. After approximately 100 molecules of thrombin bound per platelet, complete release occurred in all buffers in 2 min. The effect of anions was specific for the thrombin-receptor interaction as there was no corresponding effect on the binding of erythroagglutinating phytohemagglutinin (E-PHA) to platelets nor on E-PHA or collagen-induced serotonin release. The various anions did not alter platelet morphology as judged by electron microscopy. The anions had no effect on thrombin esterase catalytic activity. In addition, the total number of thrombin receptors per platelet was approximately the same in all buffers. Thus anions alter the affinity between platelet thrombin receptors and a site on thrombin distinct from the catalytic site. We conclude that the thrombin receptor is essential for thrombin-induced platelet reactions.